The Renilla Luciferase-Modified GFP Fusion Protein is Functional in Transformed Cells

Wang, Yubao; Wang, Gefu; O’Kane, Dennis J.; Szalay, Aladar A.

doi:10.1007/978-0-585-35132-2_60

Cited by 7 publications

(13 citation statements)

References 6 publications

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“…Recombinant viruses were propagated and purified as described [16]. (MOI) 0.1, and 1.0 plaque-forming units (PFU) per cell, and at specific time points (8,12,24,48, and 72 h) were harvested and lysed. The bioluminescence of Rluc in cell extracts was measured using a ML300 microplate luminometer (Dynatech Laboratories, Inc.) and expressed as relative light units (RLU) per mg of protein.…”

Section: Construction Of Recombinant Vaccinesmentioning

confidence: 99%

Attenuation of a vaccine strain of vaccinia virus via inactivation of interferon viroceptor

Jankovics

Gridley

Fodor

et al. 2006

The Journal of Gene Medicine

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Section: Construction Of Recombinant Vaccinesmentioning

confidence: 99%

Attenuation of a vaccine strain of vaccinia virus via inactivation of interferon viroceptor

Jankovics

Gridley

Fodor

et al. 2006

The Journal of Gene Medicine

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“…An ruc-gfp fusion construct was first engineered and expressed in murine LM-TK À fibroblast cells, in embryonic stem (ES) cells and in early stage embryos by Wang et al (1996) (69). A ruc-modified gfp fusion was found to be functionally expressed in murine LM-TK À cells, whereas a reverse modified gfp-ruc fusion showed no GFP expression, probably because of misfolding (70). In 1999, Wang et al suggested that chemiluminescent energy transfer Ruc to GFP could be used to image protein-protein interactions (71).…”

Section: Fusionsmentioning

confidence: 99%

Imaging of light emission from the expression of luciferases in living cells and organisms: a review

2002

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Luciferases are enzymes that emit light in the presence of oxygen and a substrate (luciferin) and which have been used for real-time, low-light imaging of gene expression in cell cultures, individual cells, whole organisms, and transgenic organisms. Such luciferin-luciferase systems include, among others, the bacterial lux genes of terrestrial Photorhabdus luminescens and marine Vibrio harveyi bacteria, as well as eukaryotic luciferase luc and ruc genes from firefly species (Photinus) and the sea panzy (Renilla reniformis), respectively. In various vectors and in fusion constructs with other gene products such as green fluorescence protein (GFP; from the jellyfish Aequorea), luciferases have served as reporters in a number of promoter search and targeted gene expression experiments over the last two decades. Luciferase imaging has also been used to trace bacterial and viral infection in vivo and to visualize the proliferation of tumour cells in animal models.

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“…The ends of the larger fragment were blunted with T4 DNA polymerase, and the fragment was gel-purified using a Geneclean kit (Qbiogene, Carlsbad, Calif.). In a parallel reaction, pCEP-4-ruc-gfp (Wang et al 1997) was double-digested with SalI and KpnI, and blunted with T4 DNA polymerase. The smaller fragment containing the ruc-gfp sequence was similarly purified.…”

Section: Construction Of Plasmid Vectorsmentioning

confidence: 99%

“…Nevertheless, since an external substrate is not required for GFP fluorescence in living tissues, GFP would be potentially advantageous for studying gene expression in living organisms. The Ruc-GFP fusion protein has been shown to retain both luciferase and GFP functions in cell cultures (Wang et al 1997;Liu et al 2000). We have also employed the Ruc-GFP fusion protein as a marker to monitor gene activation in real time in living cells and animals, and to locate GFP coexpression in individual cells (see the previous paper).…”

mentioning

confidence: 97%

A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo

Szalay

2002

Mol Gen Genomics

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In this study, we used a steroid-induced promoter activation system as a molecular switch to study the exogenous activation of transgene expression. This promoter activation system consists of three components: (1) a steroidal inducer drug, mifepristone (RU486), which binds to (2) a chimeric transcription factor complex, consisting of the mutant human progesterone receptor fused to the yeast GAL4 DNA-binding domain and the activation domain of the herpes simplex virus protein VP16, and (3) a synthetic promoter, consisting of a series of GAL4 recognition sequences upstream of the adenovirus major late E1B TATA box, linked to a gene construct (ruc-gfp) encoding a Renilla luciferase- Aequorea green fluorescent protein (GFP) fusion protein. Transcription of the promoter-marker gene cassette is activated by the drug (mifepristone)-bound chimeric transcription factor complex. Monitoring of induced gene expression was carried out using a low-light video camera and a UV microscope to detect luciferase and GFP, respectively. Using this activation system, we observed a 10- to 25-fold activation, depending on the inducer dose, of both luciferase and GFP expression in transiently transfected cells in comparison to cells that were not exposed to mifepristone. We further demonstrated activation of gene expression from the promoter activation system in live animals. The plasmids PAP CMV-GL914VPc'SV, carrying the chimeric transcription factor cassette, and plasmid p17x4-TATA-ruc-gfp, carrying the ruc-gfp reporter gene construct, were co-injected into limb muscles of nude mice. Following DNA injection, mifepristone (50 micro g/kg) was delivered by intraperitoneal injection. Thirty-six hours after DNA and mifepristone injection, significant Renilla luciferase activity was detectable in the limb muscles. The promoter activation system was also demonstrated in limb muscles and livers of nude mice that had received transplants of ex vivo-modified cells, which were transiently transformed with both the chimeric activator plasmid and the ruc-gfp reporter plasmid prior to implantation. Significant Renilla activity and GFP fluorescence were detected externally in limb muscles and in the livers of anesthetized animals that had received an intraperitoneal injection of inducer. This external monitoring method for observing inducible gene expression in live animals will facilitate experimental studies of fundamental questions of biological and therapeutic relevance. It will be especially valuable for the analysis of gene function at specific stages of animal development. The method should also be of general use in gene therapy, since it permits simultaneous monitoring of the expression levels of light-emitting proteins and therapeutic proteins originating from the activation of identical promoters.

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The Renilla Luciferase-Modified GFP Fusion Protein is Functional in Transformed Cells

Cited by 7 publications

References 6 publications

Attenuation of a vaccine strain of vaccinia virus via inactivation of interferon viroceptor

Attenuation of a vaccine strain of vaccinia virus via inactivation of interferon viroceptor

Imaging of light emission from the expression of luciferases in living cells and organisms: a review

A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo

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